The use of fluorescent dyes as detection labels has found widespread use in molecular biology, cell biology and molecular genetics. For example, the use of fluorescently-labeled oligonucleotides is now widespread in a variety of different assays, including polynucleotide sequencing, fluorescence in situ hybridization (FISH), hybridization assays on nucleic acid arrays, fluorescence polarization studies, and nucleic acid amplification assays, including polymerase chain amplification assays carried out with fluorescent probes and/or primers.
Some fluorescent labels can be attached to nascent or completed oligonucleotide chains synthesized in situ using fluorescent phosphoramidite reagents. For example, fluorescein phosphoramidite reagents are available commercially (see, e.g., 2006 product catalog of Glen Research Corporation, Sterling, Va.). In such reagents, the 3′- and 6′-exocyclic oxygen atoms of the fluorescein ring are protected with pivaloyl groups to prevent side reactions. Modification of the fluorescien ring with these groups also holds the carboxylate group at the 3-position in the closed, spiro lactone form, preventing proton donation from the carboxylate to the phosphoramidite group, which would convert this phosphoramidite group into a good leaving group, leading to decomposition of the reagent. The fluorescein ring is also stable to the conditions used to oxidize the nascent oligonucleotide and to treatment with aqueous ammonium, the standard method by which the nucleobase protecting groups are removed and the synthetic oligonucleotide is cleaved from the synthesis resin.
Unfortunately, many rhodamine dyes are susceptible to chemical modification when treated with the reagents commonly employed to oxidize and deprotect/cleave synthetic oligonucleotides negatively impacting their fluorescent properties. As a consequence, rhodamine dyes are commonly attached to oligonucleotides following synthesis, deprotection and cleavage from the synthesis resin. This adds additional steps and manual labor, resulting in greater cost and inconvenience in the overall synthesis of rhodamine-labeled oligonucleotides.
Owing to these and other limitations, there are currently only two rhodamine dyes that are commercially available as phosphoramidite reagents: tetramethyl rhodamine (“TAMRA”) and rhodamine X (“ROX”). Additional reagents that permit labeling of oligonucleotides with myriad different rhodamine dyes during solid phase chemical synthesis would be desirable.